Complex Formation and Ionic Association in Methanol Solutions of Poly(Ethylene Oxide) and Alkaline Earth Salts

Complex formation of poly(ethylene oxide) (PEO) with divalent barium and strontium salts was investigated in methanol. In these systems the complexation was accompanied by a considerable degree of ionic association. An analytical model for the polymer-ion complexation based on a one-dimensional lattice model was proposed. According to this model, the electrostatic effects between the bound ions were separated from the total free energy change of the binding. Three binding constants, i.e., the ionic association constant K _A, the cation binding constant, K _c, and the anion binding constant, K _a, could be estimated. K _A for barium and strontium salts was comparable, and the effect of counteranions on K _A was not large. K _c for barium salts was almost independent of the kind of counteranion and larger than that for corresponding strontium salts, indicating stronger polymer-ion interaction for barium salts. The anion binding constant, K _a, was strongly dependent upon the kind of anion, and the order was CI^? ? ? 4 ^?. The pronounced ion binding for larger anions may be explained by the more favorable free energy change of desolvation. Finally, the concentration of free and bound ionic species was determined as a function of PEO concentration.     

Oil-Water Partitioning of a Synthetic Tetracarboxylic Acid as a Function of pH

The oil-water partitioning of a synthetic tetraacid acting as a model compound for indigenous C₈₀-C₈₂ ARN acids has been studied as a function of pH, ionic strength and type of monovalent counterion. Experimental data obtained with ultraviolet-visible and HPLC/UV analyses have been fitted to thermodynamic models based on one, two or four dissociation steps to obtain o/w partition coefficients (K _wo ) of the fully protonated acid between chloroform and aqueous solutions, and its apparent acidity constant(s), pK _a. As the study is conducted above the CMC of the tetraacid, in general high apparent acidity constants were obtained in the range from 6 to 8 resulting from micellization equilibria. K _wo values were obtained in the range from 10^?3 to 10^?4, and decreasing with increasing salinity. At 50 mM K⁺, no conclusions could be made regarding the number of distinguishable dissociation steps, while at higher ionic strength (184 mM and 452 mM K⁺) and at 184 mM Na⁺ a model with two dissociation steps provided good fits to the experimental data. The first step was found to be given by a pK _a ≈ 6.6–6.8 and the second dissociation step at pK _a values ≈ 7.8–8.3. The two-step mechanism supports previous results obtained by potentiometric titrations. No significant difference in the o/w behavior was observed when changing the counterion from potassium to sodium. The main partitioning of the tetraacid in the aqueous phase occurred above pH 8, where the fully deprotonated acid was formed.     

Study on the Interaction of Raloxifene and Bovine Serum Albumin by the Tachiya Model

The interaction of bovine serum albumin (BSA) with raloxifene was assessed via fluorescence spectroscopy. The number of binding sites and the apparent binding constants between raloxifene and BSA were analyzed using the Tachiya model and Stern-Volmer equation, respectively. The apparent binding constant and the number of binding sites at 298 K were 2.33×10⁵ L?mol^?1 and 1.0688 as obtained from the Stern-Volmer equation and 2.00×10⁵ L?mol^?1 and 2.6667 from the Tachiya model. The thermodynamic parameters ΔH and ΔS were calculated to be 69.46 kJ?mol^?1 and 121.12 J?K^?1?mol^?1, respectively, suggesting that the force acting between raloxifene and BSA was mainly a hydrophobic interaction. The binding distance between the donor (BSA) and acceptor (raloxifene) was 4.77 nm according to Förster’s nonradiational energy transfer theory. It was also found that common metal ions such as K⁺, Cu²⁺, Zn²⁺, Mg²⁺ and Ca²⁺ decreased the apparent association constant and the number of binding sites between raloxifene and BSA.     

Comparing the Affinities of Flavonoid Isomers with Protein by Fluorescence Spectroscopy

Abstract

Dietary flavonoids can be detected in plasma as protein‐bound conjugates. Flavonoids–protein interaction is expected to modulate the bioavailability of flavonoids. In this work, the binding flavonoid isomers (galangin, baicalein, apigenin, and genistein; MW=270.25) and B‐ring hydroxylation flavonols (galangin, kaempferol, quercetin, and myricetin, which share the same structure on the A and C rings but have 0, 1, 2, and 3 moieties of ‐OH on the B‐ring, respectively) to protein were investigated by fluorescence quenching method. The apparent binding constants (K _a ) of were flavonoid isomers determined as: flavones (10⁶–10⁷ L mol^?1)>isoflavone≈flavonol (10⁵ L mol^?1). For B‐ring hydroxylation flavonols, the binding affinity increased with increasing number of hydroxyl groups on the B‐ring. The binding constants (K _a ) were determined as follows: myricetin>quercetin>kaempferol>galangin.     

Some General Features in the Autocatalytic Reaction between Sulfite Ion and Different Oxidants

The autocatalytic oxidation of a weak acid is a common building block of the pH oscillators. These reactions can be described by a simple general scheme that includes a protonation equilibrium and the oxidation of the protonated form of the weak acid. Here we show that independently from the chemical nature of the oxidizing agent, these reactions bear some general features, namely (1) the change in pH (ΔpH) observed during the reaction is determined by the acidity constant (K_HA) and by the initial concentration of the unprotonated form of the weak acid (A^?): , (2) the inflection time of the autocatalytic reaction (t_i) depends reciprocally on K_HA and on the initial hydrogen ion concentration, and (3) in the presence of a competitive reversible proton‐binding component (D^?), that is not involved in the oxidation process, ΔpH follows a titration‐like curve as the concentration of D^? is increased, t_i is only slightly affected but the maximum rate of the autocatalytic process is significantly reduced. The slowing of the overall reaction is proportional to the acidity constant of the proton‐binding component.     

2‐(2,4‐Dinitrobenzyl)pyridine (DNBP): A Potential Light‐Activated Proton Shuttle

The well‐known photochromic tautomerism of 2‐(2,4‐dinitrobenzyl)pyridine ( 1 ; CH; Scheme 1) was re‐investigated by flash photolysis in aqueous solution in view of its potential application as a light‐activated proton pump. Irradiation of 1 yields the enamine tautomer NH (λ_max=520 nm) that rapidly equilibrates with its conjugate base CNO^? (λ_max=420 nm). The pH–rate profile for the first‐order decay of NH and CNO^? provides a direct determination of the acidity constant of NH, pK =5.94±0.12 (I=0.1M ) and serves to clarify the mechanisms of proton transfer prevailing in aqueous solutions. The acidity constant of protonated 1 (CHNH⁺), pK =4.18±0.02, was determined by spectrophotometric titration.     

Study of Activation and Inhibition of Certain Metal Ions to Amylase Catalyzed Reaction by Microcalorimetry

IntroductionMostcomplicatedreactionshappenedinlivingcrea tures ,amongthemenzymecatalyzedreactionisanimpor tantclass .Itissignificantinboththeoryandpracticetoinvestigateenzymecatalyzedreaction .Therearemanyex perimentalmethodssuchasspectrophotometry ,titrimetry ,isotopemethod ,microcalorimetryandsoon ,inwhichmi crocalorimetryisanewoneduetoitshighsensitivityandaccuracy .Wecanstudythewholeprocessoftheheatef fectusingamicrocalorimeter .Sincetheabsorptionorpro ductionofheatisanintrinsicpropertyofe…     

ACE applied to the quantitative characterization of benzo‐18‐crown‐6‐ether binding with alkali metal ions in a methanol–water solvent system

ACE was applied to the quantitative evaluation of noncovalent binding interactions between benzo‐18‐crown‐6‐ether (B18C6) and several alkali metal ions, Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺, in a mixed binary solvent system, methanol–water (50/50 v/v). The apparent binding (stability) constants (K_b) of B18C6–alkali metal ion complexes in the hydro‐organic medium above were determined from the dependence of the effective electrophoretic mobility of B18C6 on the concentration of alkali metal ions in the BGE using a nonlinear regression analysis. Before regression analysis, the mobilities measured by ACE at ambient temperature and variable ionic strength of the BGE were corrected by a new procedure to the reference temperature, 25°C, and the constant ionic strength, 10 mM . In the 50% v/v methanol–water solvent system, like in pure methanol, B18C6 formed the strongest complex with potassium ion (log K_b=2.89±0.17), the weakest complex with cesium ion (log K_b=2.04±0.20), and no complexation was observed between B18C6 and the lithium ion. In the mixed methanol–water solvent system, the binding constants of the complexes above were found to be about two orders lower than in methanol and about one order higher than in water.     

IONIC STRENGTH EFFECTS ON THE GROUND STATE COMPLEXATION and TRIPLET STATE ELECTRON TRANSFER REACTION BETWEEN ROSE BENGAL and METHYL VIOLOGEN

Abstract— The equilibrium constants, K_c, for complexation between methyl viologen dication (MV²+) and Rose Bengal, or Eosin Y, decrease with increasing ionic strength. At zero ionic strength K_c is 6500 (± 500) mol^?1 dm³ for Rose Bengal and 3200 (± 200) mol^?1 dm³ for Eosin Y, and these values decrease to 1500 (± 100) and 680 (± 40) mol^?1 dm³, respectively, at an ionic strength of 0.1 mol dm^?3. K_c is independent of pH between 4.5 and 10. ΔH is -25 (± 1) kJ mol^?1 for complexation with either dye, whereas ΔS is -15 (± 3) J K^?1 mol^?1 for Rose Bengal, and - 23 (± 3) J K^?1 mol^?1 for Eosin Y. The complexation constant for Rose Bengal and the neutral viologen, 4,4'-bipyridinium-N, N'-di(propylsulphonate), (4,4'-BPS), is 420 (± 35) mol^?1 dm³, and independent of ionic strength. No complexation could be observed for either Rose Bengal or Eosin with another neutral viologen, 2,2'-bipyridinium-N,N'-di(propylsulphonate), (2,2'-BPS). MV²+ quenches the triplet state of Rose Bengal with a rate constant of 7 × 10⁹ mol^?1 dm³ s^?1, and this rate constant decreases slightly as ionic strength increases. The cage escape yield following quenching, Φ_cc is very low (Φ_cc= 0.02 (± 0.005), and independent of ionic strength. 4,4'-BPS quenches the triplet state of Rose Bengal with a rate constant of 2.2 (± 0.1) × 10⁹ mol^?1 dm³ s^?1, and gives a cage escape yield of 0.033 (± 0.006). 2,2'-BPS quenches the Rose Bengal triplet with a rate constant of 6 (± 1) × 10⁸ mol^?1 dm³ s^?1 and gives a cage escape yield of 0.07 (± 0.01). Conductivity measurements indicate that MV²+(Cl^?)₂ is completely dissociated at concentrations below 2 × 10^?2 mol dm^?3.     

Kinetics and mechanisms of oxidation by metal ions,part XII [1] oxidation of formaldehyde by alkaline osmium(VIII)

The stopped-flow measurements on the disappearance of alkaline osmium(VIII) at 402 nm indicated a first-order dependence each in [Os(VIII)] and [HCHO]. The pseudo first-order rate constant k_obs ([HCHO] ? [Os(VIII)]) decreased with increasing [OH^?]. The ionic strength, however, had no effect on k_obs. The rapid scan spectra of the reaction mixture indicated the formation of an inert complex which absorbs at 319 nm. Therefore the rate determining step is considered to involve the bimolecular collision between OsO₄(OH) and hydrated formaldehyde. The values of the rate limiting constant k and the equilibrium constant K_ha for the formation of the alkoxide ion from the reaction of hydrated formaldehyde with OH^? are evaluated. The equilibrium constant K_ha, within the experimental limits, is independent of temperature. The pK_a value, calculated from K_ha, is 13.62 ± 0.05 which is in good agreement with the pK_a value 13.27 for formaldehyde. The activation parameters, ΔH? = 40 ± 2 kJ mol^?1 and ΔS? = ?51 ± 6 JK^?1 mol^?1, for the rate limiting constant k are determined.     

Determination of acidity constants,ionic mobilities,and hydrodynamic radii of carborane-based inhibitors of carbonic anhydrases by capillary electrophoresis

Capillary electrophoresis (CE) has been applied for determination of the thermodynamic acidity constants (pK_a) of the sulfamidoalkyl and sulfonamidoalkyl groups, the actual and limiting ionic mobilities and hydrodynamic radii of important compounds, eight carborane-based inhibitors of carbonic anhydrases, which are potential new anticancer drugs. Two types of carboranes were investigated, (i) icosahedral cobalt bis(dicarbollide)(1-) ion with sulfamidoalkyl moieties, and (ii) 7,8-nido-dicarbaundecaborate with sulfonamidoalkyl side chains. First, the mixed acidity constants, pK_a^mix, of the sulfamidoalkyl and sulfonamidoalkyl groups of the above carboranes and their actual ionic mobilities were determined by nonlinear regression analysis of the pH dependences of their effective electrophoretic mobility measured by capillary electrophoresis in the pH range 8.00−12.25, at constant ionic strength (25 mM), and constant temperature (25°C). Second, the pK_a^mix were recalculated to the thermodynamic pK_as using the Debye–Hückel theory. The sulfamidoalkyl and sulfonamidoalkyl groups were found to be very weakly acidic with the pK_as in the range 10.78−11.45 depending on the type of carborane cluster and on the position and length of the alkyl chain on the carborane scaffold. These pK_as were in a good agreement with the pK_as (10.67−11.27) obtained by new program AnglerFish (freeware at https://echmet.natur.cuni.cz ), which provides thermodynamic pK_as and limiting ionic mobilities directly from the raw CE data. The absolute values of the limiting ionic mobilities of univalent and divalent carborane anions were in the range 18.3−27.8 TU (Tiselius unit, 1 × 10⁻⁹ m²/Vs), and 36.4−45.9 TU, respectively. The Stokes hydrodynamic radii of univalent and divalent carborane anions varied in the range 0.34−0.52 and 0.42−0.52 nm, respectively.     

Estimation of acidity constants,ionic mobilities and charges of antimicrobial peptides by capillary electrophoresis

Capillary electrophoresis (CE) was employed for the determination of thermodynamic acidity constants (pK_a) and actual ionic mobilities of polycationic antimicrobial peptides (AMPs). The effective electrophoretic mobilities of AMPs were measured by CE in a series of the background electrolytes within a wide pH range (2.00–12.25), at constant ionic strength (25 mM) and ambient temperature, using polybrene coated fused silica capillaries to suppress sorption of cationic AMPs to the capillary wall. Eventually, Haarhoff–Van der Linde peak fitting function was used for the determination of correct migration times of some AMPs peaks that were distorted by electromigration dispersion. The measured effective mobilities were corrected to 25°C. Mixed acidity constants, , and actual ionic mobilities, m_i, of AMPs were determined by the nonlinear regression analysis of pH dependence of their effective mobilities. The values were recalculated to thermodynamic pK_as using the Debye–Hückel theory. Thermodynamic pK_a of imidazolium group of histidine residues was found to be in the range 3.72–4.98, pK_a of α‐NH₃⁺ group was in the range 6.14–6.93, and pK_a of ε‐NH₃⁺ group of lysine spanned the interval 7.26–9.84, depending on the particular amino acid sequence of the AMPs. Actual ionic mobilities of AMPs with positive charges from one to six elementary units achieved values (9.8 – 36.5) × 10^?9 m²V^?1s^?1.     

Phase-dependent photochromism of a lactone-stabilized chromene from a flavylium reaction network

New trans‐2‐hydroxychalcones bearing a carboxylate group at position 2′ ( Ct ^?) were synthesized (compounds 2 and 3 ). These compounds lead to a network of chemical reactions depending on pH value, light, and solvent. In water, when the pH value is lowered, the ionized trans‐chalcone is protonated and the flavylium cation A H⁺ is formed at very acidic pH values through hemiketal B and cis‐chalcone Cc , with global acidity constants of pK′_a ≤?1 and ≈0.1, respectively, for 2 and 3 . The electron‐acceptor character of the carboxylic substituent not only increases the observed acidity of the flavylium cation, but also decreases the rate of the ring‐opening/‐closing from a subsecond timescale to hours relative to model compound 1 (without carboxylate). The photochemistry of the network was studied in detail by means of continuous irradiation, monitored by UV/Vis absorption and ¹H and ¹³C NMR spectroscopic analysis. Although compound 3 is only slightly photoactive, compound 2 ( Ct^? ) reacts in aqueous solutions (λ_irr=313 nm) to form B^? and Cc^? , with a global quantum yield of 0.15, and fully reverts back to Ct^? with a rate constant of k=6.7×10^?5 s^?1. The flavylium cation is no longer formed in methanol, and irradiation of Ct^? leads to the formation of B ^? and the new lactone‐trapped chromene species La . The formation of La takes place through a sequence of three photochemical steps: photoisomerization of Ct ^?, photo‐ring‐closing reaction of Cc ^?, and photolactonization of B ^?. Only the cis/trans isomerization and ring‐closing reactions are thermally reversible on a timescale of seconds and hours, respectively. A photochromic system was achieved in rigid matrices of methanol (at 77 K) and 1‐dodecanol (5 °C) by irradiating lactone La to give a red ortho‐quinone allide through a photo‐ring‐opening reaction; the color disappears with a rate constant of k=1.25×10^?2 s^?1 in 1‐dodecanol at 5 °C.     

Microspheres Sensor Based on Molecularly Imprinted Polymer Synthesized by Precipitation Polymerization

IntroductionSincethepiezoelectricbulkacousticwave (BAW)sen sorswereappliedinliquidphaseinthe 1980s ,manypapershavebeenreported .1,2 However,theapplicationofBAWsen sorsbasedonmasseffectislimitedbecauseoflackofthespecialselectivitytotheanalyte.Variousmethodshavebeenproposedtosolvethisproblem ,especiallytheapplicationofthebiomaterials .3Unfortunately ,theresultwasnotsogoodasexpected,duetotheinstinctdisadvantageofthebiomateri als ,e.g .,poorstability ,shortlifespan ,althoughpossess inghighselec…     

Spectrophotometric investigation of monobromo and dibromo complexes of copper(II) in methanolic medium

Complex formation between copper(II) and bromide in anhydrous methanol was investigated spectrophotometrically. At a constant copper(II) concentration of 3.0 x 10^?4M, Cu²⁺ and CuBr⁺ are at equilibrium for [Br^?] < 1.0 x 10^?3M while CuBr⁺ and CuBr₂ exist at equilibrium in the range of [Br^?] 2.0 x 10^?3 ?40 x 10^?3M. An isosbestic point at 235 nm indicated the equilibrium of Cu²⁺ and CuBr⁺ while a second isosbestic point at 290 nm showed the equilibrium of CuBr⁺ and CuBr₂. Stability constants for the formation of CuBr⁺ and CuBr₂ (K₁, and K₂, respectively) were determined as a function of ionic strength in the range 0.01–0.10. Log K₁ and log K₂ values at zero ionic strength were obtained by extrapolation of the plot of log K vs ionic strength, the values being 3.97 ± 0.01 and 2.31 ± 0.01.     

Spectroscopy studies on DNA binding of two ruthenium complexes [Ru(MeIm)4(L)]2+ (L = iip and tip)

Two ruthenium(II) complexes [Ru(MeIm)₄(L)]²⁺ (L?=?2-(imidazo-4-group)-1H-imidazo-[4,5-f][1,10]phenanthroline, 2-(thiophene-2-group)-1H-imidazo[4,5-f][1,10]phenanthroline, MeIm?=?1-methylimidazole) have been synthesized according to literature and structurally characterized. The interaction of the complexes with calf thymus DNA has been explored using electronic absorption titration, competitive binding experiment, circular dichroism, thermal denaturation, and viscosity measurements. The results show that both complexes could bind DNA in a intercalation mode and the DNA-binding affinity of [Ru(MeIm)₄(tip)]²⁺ (K _b?=?(7.2?±?0.3)?×?10⁵?(mol?L^?1)^?1) is greater than that of [Ru(MeIm)₄(iip)]²⁺ (K _b?=?(6.1?±?0.2)?×?10⁵?(mol?L^?1)^?1).     

Synthesis,structure, spectroscopic,and DNA binding properties of Co(III) and Ni(II) complexes with ((E)-2-(amino((pyridin-2-ylmethylene)amino)methylene)maleonitrile)

Two new complexes, [Co(L)₂]Cl·(MeOH)₂ (1) and [Ni(L)₂]₄·EtOH (2) (L?=?(E)-2-(amino((pyridin-2-ylmethylene)amino)methylene)maleonitrile), were synthesized and characterized by X-ray crystallography, IR, UV, and fluorescence spectroscopy. According to X-ray crystallographic studies, each metal was six-coordinate with six nitrogens from two ligands. Both complexes form two-dimensional supramolecular networks via hydrogen bonding and π–π interactions. Ultraviolet and visible spectra showed that absorptions arise from π–π ^?, MLCT, and d–d electron transitions. Fluorescence spectroscopy revealed moderate intercalative binding of these two complexes with EB–DNA, with apparent binding constant (K _app) values of 9.14?×?10⁵ and 3.20?×?10^5?M^?1 for Co(III) and Ni(II) complexes, respectively. UV–visible absorption spectra showed that the absorption of DNA at 260?nm was quenched for 2 but quenched then improved for 1 with addition of complexes, tentatively attributed to the effect of the combined intercalative binding and electrostatic interaction for 1.     

Study of solvent effects on the stability constant and ionic mobility of the dibenzo‐18‐crown‐6 complex with potassium ion by affinity capillary electrophoresis

The effect of solvent on the strength of noncovalent interactions and ionic mobility of the dibenzo‐18‐crown‐6 complex with K⁺ in water/organic solvents was investigated by using affinity capillary electrophoresis. The proportion of organic solvent (methanol, ethanol, propan‐2‐ol, and acetonitrile) in the mixtures ranged from 0 to 100 vol.%. The stability constant, K_KL, and actual ionic mobility of the dibenzo‐18‐crown‐6‐K⁺ complex were determined by the nonlinear regression analysis of the dependence of the effective electrophoretic mobility of dibenzo‐18‐crown‐6 on the concentration of K⁺ (added as KCl) in the background electrolyte (25 mM lithium acetate, pH 5.5, in the above mixed hydro–organic solvents). Competitive interaction of the dibenzo‐18‐crown‐6 with Li⁺ was observed and quantified in mixtures containing more than 60 vol.% of the organic solvent. However, the stability constant of the dibenzo‐18‐crown‐6‐Li⁺ complex was in all cases lower than 0.5 % of K_KL. The log K_KL increased approximately linearly in the range 1.62–4.98 with the increasing molar fraction of organic solvent in the above mixed solvents and with similar slopes for all four organic solvents used in this study. The ionic mobilities of the dibenzo‐18‐crown‐6‐K⁺ complex were in the range (6.1–43.4) × 10^?9 m² V^?1 s^?1.     

A kinetic study of the oxidation of L-ascorbic acid by chromium(VI)

The reaction between chromium(VI) and L-ascorbic acid has been studied by spectrophotometry in the presence of aqueous citrate buffers in the pH range 5.69–7.21. The reaction is slowed down by an increase of the ionic strength. At constant ionic strength, manganese(II) ion does not exert any appreciable inhibition effect on the reaction rate. The rate law found is where K_p is the equilibrium constant for protonation of chromate ion and k_r is the rate constant for the redox reaction between the active forms of the oxidant (hydrogenchromate ion) and the reductant (L-hydrogenascorbate ion). The activation parameters associated with rate constant k_r are E_a = 20.4 ± 0.9 kJ mol^?1, ΔH^≠ = 17.9 ± 0.9 kJ mol^?1, and ΔS^≠=?152 ± 3 J K^?1 mol^?1. The reaction thermodynamic magnitudes associated with equilibrium constant K_p are ΔH⁰ = 16.5 ± 1.1 kJ mol^?1 and ΔS⁰ = 167 ± 4 J K^?1 mol^?1. A mechanism in accordance with the experimental data is proposed for the reaction. © 1993 John Wiley & Sons, Inc.     

Fabrication and Electrochemical Behavior of Hemoglobin Modified Carbon Ionic Liquid Electrode

A new hemoglobin (Hb) and room temperature ionic liquid modified carbon paste electrode was constructed by mixing Hb with 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMIMPF₆) and graphite powder together. The Hb modified carbon ionic liquid electrode (Hb‐CILE) was further characterized by FT‐IR spectra, scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). Hb in the carbon ionic liquid electrode remained its natural structure and showed good direct electrochemical behaviors. A pair of well‐defined quasireversible redox peaks appeared with the apparent standard potential (E′) as ?0.334 (vs. SCE) in pH 7.0 phosphate buffer solution (PBS). The electrochemical parameters such as the electron transfer number (n), the electron transfer coefficient (α) and the heterogeneous electron transfer kinetic constant (k_s) of the electrode reaction were calculated with the results as 1.2, 0.465 and 0.434 s^?1, respectively. The fabricated Hb‐CILE exhibited excellent electrocatalytic activity to the reduction of H₂O₂. The calibration range for H₂O₂ quantitation was between 8.0×10^?6 mol/L and 2.8×10^?4 mol/L with the linear regression equation as I_ss (μA)=0.12 C (μmol/L)+0.73 (n=18, γ=0.997) and the detection limit as 1.0×10^?6 mol/L (3σ). The apparent Michaelis–Menten constant (K_M^app) of Hb in the modified electrode was estimated to be 1.103 mmol/L. The surface of this electrochemical sensor can be renewed by a simple polishing step and showed good reproducibility.